Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule

Abstract

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Magnetized chitosan hydrogel and silk fibroin, reinforced with PVA: a novel nanobiocomposite for biomedical and hyperthermia applications

“…Eventually, the supernatants were transferred to the flat bottom plate and the OD of each was determined at 405 nm using the ELISA reader (Biohit, Finland). 36,37 The hemolysis ratio was calculated by following formula: 38 Hemolysis ratio (HR) = (TS − NC)/(PC − NC) × 100TS = test sample, NC = negative control, PC = positive control.…”

Decoration of graphene oxide nanosheets with carboxymethylcellulose hydrogel, silk fibroin and magnetic nanoparticles for biomedical and hyperthermia applications

“…The supernatant of each well was transferred to the flat bottom plate and OD was quantified using an ELISA reader (Biohit, Finland) at 405 nm. 35 …”

“…The plate was incubated for 2 h at 37 °C and then centrifuged at 2000 rpm for 10 min. The supernatant of each well was transferred to the flat bottom plate and OD was quantified by the ELISA reader (Biohit, Finland) at 405 nm 35 .…”

Agar-tragacanth/silk fibroin hydrogel containing Zn-based MOF as a novel nanobiocomposite with biological activity

“…Supernatant photometric analysis was performed to quantify the absorbance values at a wavelength of 405 nm using an ELISA Reader. The hemolysis percentage of the samples was determined utilizing the following formula 43 : …”